A vigorous angiogenic response is a prominent component of normal wound repair, and a long-standing assumption is that robust angiogenesis is required for optimal healing. During the last funding period, our laboratory established that angiogenesis is reduced in wounds that heal scarlessly or with minimal scar formation, including wounds in fetal skin and oral mucosa. Compared to wounds of adult skin, both fetal and mucosal wounds contain reduced levels of the proangiogenic factor vascular endothelial cell growth factor (VEGF), and exhibit a significant decrease in vascular growth over baseline. Within wounds, keratinocytes are known to be a primary source of VEGF, and our preliminary studies suggest that skin keratinocytes produce more VEGF than keratinocytes from oral mucosa. Thus the keratinocyte response might dictate the angiogenic response in wounds. The long term objective of this research is to understand the regulation of angiogenesis in wounds. The central hypothesis of the current application is that the differential patterns of wound angiogenesis that occur amongst adult skin, fetal skin, and adult mucosa influence scar formation and are derived from intrinsic differences in the keratinocyte response to injury at each site. Our experimental plan takes advantage of multiple model systems, both in mice and humans. Aim 1 will determine if wound angiogenesis is mechanistically different in murine adult skin, mucosa, and fetal skin. A VEGF-lacZ transgenic system will be used to learn how VEGF production in wounds differs among the three sites. The levels of factors that can regulate VEGF and angiogenesis, including hypoxia and hypoxia- inducible factor (HIF), will also be quantified. Parallel studies in humans will examine the angiogenic response in paired mucosal and skin wounds. Aim 2 will discern how the regulation of VEGF production differs in keratinocytes from mucosa and adult skin. In vitro experiments will compare the production of VEGF in mucosal and skin keratinocytes when exposed to varying levels of hypoxia. Mechanistic experiments will utilize an organotypic culture to examine the influence of fibroblasts on the keratinocyte phenotype. In Aim 3, we will determine how VEGF production influences the production of TGFpl, a known pro-fibrotic factor, in the context of the healing wound. In other experiments, we will manipulate the angiogenic response to determine if robust angiogenesis is mechanistically linked to scar formation. The results of these studies will improve our understanding of site specific differences in the response of keratinocytes to injury. Ultimately, our findings may assist in the development of strategies to improve outcomes for the estimated one million Americans who suffer from problems with healing wounds.